Microbial pathogenes which infect mucosal surfaces of the body continue to cause significant human morbidity and mortality. Current parenteral vaccination strategies for these infections are frequently ineffective. Antigens in the lumen of the gastrointestinal tract bind to specialized M cells in the epithelium and are presented to underlying cells of the immune system, leading to proliferation and differentiation of IgA- committed, antigen-specific lymphocytes which circulate to the lamina propria of diverse mucosal surfaces. This dissemination of a mucosal immune response from an individual inductive site to diverse effector sites has been termed the common mucosal immune system. Vibrio cholerae is an excellent model for studying mucosal immunity and stimulation of a common mucosal immune response. V. cholerae selectively adheres to M cells of the gastrointestinal tract and natural infection with V. cholerae is followed by long-lasting, systemic and mucosal immune responses. V. cholerae also has many advantages as a vector system for delivering antigens from heterologous organisms to the mucosal immune system. The long-term goal of these studies is to develop V. cholerae as a live, oral, attenuated vaccine vector strain for delivery of heterologous antigens to the mucosal immune system. There are four SPECIFIC AIMS in the present proposal: (1) optimizing in vivo expression of heterologous antigens by live, oral, attenuated vector strains of V. cholerae, using the B subunit of cholera toxin (CTxB) as a model antigen; (2) analysis of a fusion protein between the serine rich protein of Entamoeba histolytica (SREHP) and CtxB, expressed by a live, oral, attenuated V. cholerae vector, for inducing anti-SREHP mucosal immunity in an animal model; (3) examining the role of mutant LT-I as an immunoadjuvant, when expressed by live, oral, attenuated V. cholerae vector strains; and (4) use of live, oral, attenuated V. cholerae vector strains to deliver epitopes of toxin A of Clostridium difficile: application of general principles to a specific mucosal infection and assessment of the role of antigenic context in the mucosal immune response.